A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts.
A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts.
复制标题
DOI：
10.1007/s11306-019-1544-z
复制
发表时间：
2019-05-23
期刊：
影响因子：
--
通讯作者：
Trushina E
中科院分区：
文献类型：
--
作者：
Wilkins J;Sakrikar D;Petterson XM;Lanza IR;Trushina E
患者来源的皮肤成纤维细胞为研究多种人类疾病的分子机制提供了独特的翻译模型。代谢组学图谱可以跟踪一系列代谢物和相互关联的代谢途径的变化，从而了解疾病发生和发展的分子机制，以及治疗干预的效果。因此，建立标准化的人体皮肤成纤维细胞代谢组学分析方案对严格和可靠的代谢评估是重要的。我们旨在开发一种优化的方法，用于同时测定人皮肤成纤维细胞中氨基酸、酰肉碱和三羧酸(TCA)循环成分的浓度，采用气相色谱(GC)和液相色谱(LC)与质谱仪(MS)相结合的方法。利用GC/MS和LC/MS分析平台，确定了四种不同的细胞收获方法对氨基酸、酰肉碱和三氯乙酸循环代谢物回收的适用性。每种方法在−80°C保存48小时、2周和1个月后测定代谢物的稳定性。在80%甲醇溶液中收获细胞可以获得最好的代谢物回收和保存。在−80°C下，样品在80%甲醇中储存长达一个月，对代谢物浓度没有显著影响。我们开发了一种用于人皮肤成纤维细胞代谢组学分析的可靠工作流程，适用于高通量多平台分析。这种方法允许对在不同时间收集的样本中的代谢变化进行直接的并排比较，这些样本可以用于大型患者队列的研究。
Patient-derived skin fibroblasts offer a unique translational model to study molecular mechanisms of multiple human diseases. Metabolomics profiling allows to track changes in a broad range of metabolites and interconnected metabolic pathways that could inform on molecular mechanisms involved in disease development and progression, and on the efficacy of therapeutic interventions. Therefore, it is important to establish standardized protocols for metabolomics analysis in human skin fibroblasts for rigorous and reliable metabolic assessment. We aimed to develop an optimized protocol for concurrent measure of the concentration of amino acids, acylcarnitines, and components of the tricarboxylic acid (TCA) cycle in human skin fibroblasts using gas (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS). The suitability of four different methods of cell harvesting on the recovery of amino acids, acylcarnitines, and TCA cycle metabolites was established using GC/MS and LC/MS analytical platforms. For each method, metabolite stability was determined after 48 hours, two weeks and one month of storage at −80°C. Harvesting cells in 80% methanol solution allowed the best recovery and preservation of metabolites. Storage of samples in 80% methanol up to one month at −80°C did not significantly impact metabolite concentrations. We developed a robust workflow for metabolomics analysis in human skin fibroblasts suitable for a high-throughput multiplatform analysis. This method allows a direct side-by-side comparison of metabolic changes in samples collected at different time that could be used for studies in large patient cohorts.
登录
查看更多内容
影响因子：
7.7
作者：
Lecuyer, Lucie;Bala, Agnes Victor;Touvier, Mathilde
通讯作者：
Touvier, Mathilde
影响因子：
--
作者：
Shukla, Surendra K.;Gunda, Venugopal;Singh, Pankaj K.
通讯作者：
Singh, Pankaj K.
影响因子：
14.8
作者：
Martano, Giuseppe;Delmotte, Nathanael;Vorholt, Julia A.
通讯作者：
Vorholt, Julia A.
影响因子：
3.6
作者：
Muschet, Caroline;Moeller, Gabriele;Prehn, Cornelia;de Angelis, Martin Hrabe;Adamski, Jerzy;Tokarz, Janina
通讯作者：
Tokarz, Janina
影响因子：
7.4
作者：
Luo, Xian;Li, Liang
通讯作者：
Li, Liang